Breaking down the microbiology world one bite at a time
Winning Tiny “Dirt-y” Battles
Do you remember the last time you went for a walk? No, not on concrete or in your house but on actual soil. Did you take a moment to appreciate the world blossoming beneath your feet – a world not very different from ours? The soil is like a big nation where bacteria, fungi, animals, and plants are the citizens. The waste management of the nation is performed by some bacteria and fungi called decomposers. The decomposers break down dead matter and release minerals and sources of carbon and nitrogen that all the citizens can use.
Fungi of the group Mortierella are examples of decomposers that can be found globally in diverse conditions. These fungi can simplify larger molecules into ready-to-consume sources of energy. Thus Mortierella not only prevents dead creatures from piling up but also eliminates pollutants from the soil. However, like our world, the inhabitants of dirt also form relations, some friendly and some deadly. Mortierella is preyed upon by small roundworms, also called nematodes. The fungus fights back using some powerful weapons.
In a recent publication, Hannah and her team of German and Australian scientists investigated one such possible weapon – anti nematode chemicals. These researchers were particularly interested in a specific strain of Mortierella named NRRL 6337. Two molecules belonging to the class of benzolactone enamides (Fig. 1) were extracted from the fungi. The scientists argued that these two chemicals could be cytotoxic (cytotoxic: cyto → cell; toxic → poisonous) because their structure was identical to that of two cytotoxic molecules produced by a bacterium in the group Burkholderia.
However, this revelation made the researchers question whether Mortierella was capable of synthesizing these compounds. Previous research found some individuals of the Mortierella group form an intimate relationship with symbiotic bacteria. These symbiotic bacteria live inside the fungus making this relationship an endosymbiotic one (endosymbiotic: endo → within + symbiosis → mutually benefitting relation). So who was creating the benzolactone enamides? The fungus or the bacteria?
To answer the lingering question, the scientists had to identify the endosymbiont bacteria. Some genetic studies on NRRL 6337 showed that the symbiont was a bacterium of the group Mycoavidus. Studying diverse forms of Mycoavidus belonging to different forms of Mortierella, the researchers discovered that although the presence of endosymbionts is common among Mortierella, not all Mortierella are home to the same kind of bacteria. Bacteria found within the Mortierella could have different characteristics and functional capacities. In fact, some Mortierella-Mycoavidus pairs may not even produce benzolactone enamides. Given the inconsistency, it became dubious whether the bacteria within NRRL 6337 were producing the chemicals.
The scientists grew NRRL 6337 in the presence of antibiotics, which would prevent the bacteria from growing. These fungal colonies, which were stripped of their endosymbiotic bacterial friends, did not show the presence of the cytotoxic molecules. Concluding that, the bacteria were likely the master chemists creating the chemicals.
To definitively conclude the bacteria were responsible, the scientists attempted to grow the bacteria in absence of the host fungus. However, this did not work. This explained the bacteria’s dependence on its host.
Although they could not grow the bacteria without its host, the scientists were able to achieve the difficult task of obtaining pure bacterial DNA. The sequencing led to the discovery that this bacteria was a novel individual of the group Mycoavidus called Mycoavidus necroximicus.
The genes of this symbiont were compared to those of other symbionts from different individuals of Mortierella. The comparison revealed the true chemical-producing potential of the bacteria in comparison to other related endosymbionts. A part of the genetic code of Mycoavidus necroximicus showed remarkable similarity to genes in Burkholderia B8 that were known to be responsible for the synthesis of benzolactone enamides. Finally, it was proven that this symbiotic bacteria was responsible for making the chemicals.
The final piece in the puzzle was to determine the antinematode activity of benzolactone enamides. The scientist used NRRL 6337 and grew it in two ways, one culture with the endosymbiont present and the other culture without it. Thereafter, each culture was taken and the cellular material was extracted and divided into 9 unique fractions using a method called HPLC. Thus, all 18 fractions were infected with the prototype nematode C. elegans.
If the fraction allows the growth of nematodes, it implies the absence of anti nematode activity but if the nematode fails to grow, the fraction has anti nematode potential. C. elegans was able to grow in all of the fractions from the symbiont-free fungi. However, the 6th fraction from the endosymbiont-containing NRRL 6337 possessed the ability to kill the nematodes (Fig, 2). This active fraction was studied and the benzolactone enamides were found in it.
The scientists obtained pure forms of these chemicals and determined the dose of the substance that would inhibit the growth of the nematode. To assess the validity of the finding, the experiments were performed in a nematode, A. avenae, which is a true predator of Mortierella. The study showed successful antinematodal activity of the compounds of interest.
The group of German and Australian scientists was successful in uncovering a previously unknown mechanism of microbial defense against a predatory nematode. This defense system protects the fungi that recycle dead matter, thereby enriching the soil and eventually benefiting its inhabitant. The microbial arms race has applications to our world as well. Every year countless crops are destroyed because of nematode infections. Once purified, microbial products that could kill nematodes can be created that would help farmers save their plants. Every new chemical discovery like this one is a step forward in protecting humans, our crops, and other important organisms.
The next time you walk on the soil, remember to take a moment and admire the wonders in the world beneath!
Featured image: Image Credits: Tanishq Minda
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